Structural Biochemistry/p73

p73 is part of the p53 family of transcription factors that also includes p63. The family is well known for their ability to regulate cell cycle arrest and apoptosis. p73 and p63 are both structurally and functionally similar to p53, and it is believed that they are also tumor suppressor proteins[1]. The monomer of p73 has a 50% homology with p53 causing the folding in both proteins to be very similar.[2] Even thought both proteins have a very similar structure and function, while 50% of cancers have a mutated p53 protein, but only 0.5% of cancers have a mutated p73 protein which is of great interest to scientists.[2]

The p73 protein has a DNA domain structure very similar to p53. The p73 protein is larger than the p53 protein as it has a 636 amino acid sequence.[2] Like p53, there are three structural domains that include a transactivation domain (TA) that is a proline-rich sequence following the N terminus of p73, a central DNA-binding domain (DBD) and a C terminus with an oligomerization domain (OD).[3] Because there are from the same protein family, p73 shares a 50% degree of sequence homology with p53, especially in the DBD.[2] The similarity in sequence leads to a similar folding structure which allows p73 to function in a very similar way to p53, such as recognizing and regulating targets of the p53 protein.[2] However, the oligomerization domains are less conserved between the two proteins. In this area, p73 is more similar to p63 as it can hetero-oligomerize with p63, but not with p53. [1]

Various isoforms of p73 exist. There are isoforms that posses a transactivation site similar to the site on p53 and are denoted by TAp73. Other isoforms are ΔNp73 which were originally form the TAp73 promoter transcription do not have the N-terminal transactivation domain due to aberrant splicing.[4] Studies that shown that knockout of TAp73 in mice made the mice more susceptible to carcinogens and thus more tumor-prone. However, in vivo knockout of ΔNp73 actually reduced tumor growth showing that the two different isoforms of the gene can cause different results.[4]

The p53 family proteins each work in different pathways, but they do have some overlapping functions.[2]

MDM2 degrades both p53 and p73.

p53 and p73 both have a role in triggering apoptosis

However, it has been shown that cells with only p53 knocked-out have a different chemosensitivity than cells with both p53 and p73 knocked-out[5] In chemotherapy, patients are given drugs that target fast-growing cells and kills them. Cancer cells are reproducing very quickly and thus are more likely to be the targets of these drugs, however other faster-grwoing cells such as hair and the lining of the stomach can also be targets of these drugs. In some in vivo and in vitro studies, it has been observed that mutated p53 is actually able to bind to p73 and inactivate the protein, causing the cells to be more chemoresistant. [5]

TAp73 can stop the cell cycle during the G1 and G2 phases. In the G1 phase, TAp73 arrests the cell cycle by transcriptional up-regulation of p21 and p57/Kip2 which are proteins that prevent the cell cycle from continuing.[3] In the G2 phase, p73 represses G2/M regulators that include CDC25B, CDC25C, Cyclin B1, Cyclin B2, cdc 2 and Topoisimerase IIα, thus preventing the cell from leaving the Interphase and entering Mitosis.[3] Even in mitosis, when p73 is presumed to be hyper-phosphorylated and very inactive, it still plays a role that is can transcribe p57/Kip2 genes which are negative regulators of cell proliferation.[3] TAp73 has also been shown to interact with spindle assembly checkpoints and helps with that activity and malfunction at this point is believed to be the reason for tumorigenesis.[3][4]

TAp73 can also signal for cell death to occur through various mechanisms. Through the mitochondrial pathway, TAp73 is able to up-regulate Bax and promote its movement to the mitochondria which occurs when Bax has received apoptotic signalling.[3] Through the endoplasmic reticulum stress pathway, p73 up-regulates scrotin, a gene that can induce apoptosis, in the ER as well as in supra-basal keratinocytes.[3]. It can also modulate the death receptor pathway and can induce CD95 expression (a death receptor).[3]

This isoforms of p73 is believed to be the negative of not only TAp73 but also p53 by inhibiting growth suppression and apoptosis. ΔNp73 repressed TAp73 transcipriton activity by forming inactive oligomers and competes with p53 for DNA biding sites.[3] It has been shown that in cancer cells that reductino of ΔNp73 results in apoptosis.[4] However, there is a feedback loop pathway that occurs when TAp73 and p53 are active that induces ΔNp73 creating a auto-regulatory feedback loop so that there are not too many active TAp73 and p53 molecules. It has also been shown that if DNA damage is detected, this triggers the degradation of ΔNp73, thus allowing TAp73 to function.[3]

One possible way of increasing the amount of TAp73 and decreasing the amount of ΔNp73 is by controlling the splicing of the TAp73 promoter transcripts. In hepatocellular carcinoma cells, the aberrant splicing responsible for changing the transcripts form TAp73 to ΔNp73 was caused by depletion of the splicing factor Slu7. This depletion of Slu7 was caused by c-Jun N-terminal kinase 1 (JNK-1) activity which, in turn, was caused by activation of EGFR by AR.[4] Another way of increasing the TAp73/ΔNp73 ratio to favor TAp73 is through using the COX-2 inhibitor celecoxib which has been shown to increase TAp73β in neuroblastoma cells.[4]

In most solid tumors, there is actually an abundance of p73, however it is not able to do any anti-tumor activity because it is blocked by various inhibitors that include family proteins like ΔNp73, ΔNp63, mutant p53 and non-family proteins such as MDM2, iASPP or mTOR.[4] Because of the many and complex functions of p73, it is regulated after it has already been transcribed by changing the stability or the protein, the location of the protein and the ability of the protein to bind to promoters. p73 is regulated though its degradation through the ubiquitin system.[3] MDM2 which is responsible for degrading the p53 protein can also bind to p73. However, instead of degrading the protein, it moves it to nuclear speckles and changes it's transcriptional activity. The E3 ubiquitin ligase ITCH binds to p73 to promote degradation of the protein in normal conditions. In times of stress, ITCH is downregulated and p73 and p63 levels are allowed to increase. Unlinke MDM2, ITCH is exclusive to p63 and p73.[3] Other genes that can regulate p73 include: PIAS1 that sumoylates p73 thus reducing the transcriptional activity of p73 and allowing the cell to leave the G1 phase of the cell cycle. p73's transcriptional activity is also reduced when Cyclyn/CDK complexes threonin phosphorylate it. SIRT1 inhibits the the acetylation of p73 thus decreasing the activity of p73.[3]

When normal cells were exposed to bile acids, the role of p73 was activated in response to DNA damage. Increased amounts of DNA damage due to bile acid exposure triggered only the response of p73; this result indicated that p73 induction is independent to p53 and p63, but dependent on the selective activation of c-Abl-kinase. The p73 induction activates transcription of two glycosylases, SMUG1 and MUTYH, to conduct base excision repair. When p73 is deficient, an apparent increase of DNA damage is present [6].

The causes for esophageal adenocarcinoma involve the severe progressions of gastroesophageal reflux disease, where gastric juices mix with bile acids and enter the esophagus. Exposing the esophagus to gastric acids and bile acids leads to inflammation and tissue damage. In addition, bile and acid refluxes induces genotoxic stresses, which increases ROS and RNS production and consequently causes damage to the DNA through oxidation.

Cells cultures, transfection, retroviral infections, and BA treatment
Primary fetal esophageal fibroblasts, human nontumorous esophageal epithelial cell line, and p53-null esophageal adenocarcinogen cell lines were cultured in DMEM. hTERT-immortalized esophageal epithelial EPC2 cells were grown in KSFM medium with bovine pituitary extract and epidermal growth factors. P73 was then inhibited using lentiviral transduction with shRNA or transfection of RNA, which both targeted a specific sequence found in all the isoforms.

Antibodies, immunoprecipitation, and immunofluorescence
Antibodies were used for following various proteins: p73, p63, c-Abl, phospho-c-Abl, phospho-p53, β-actin, phosphohistone H2A.X, phosphotyrosine, 8-oxoguanine, p53, p21, PUMA, SMUG1, MUTYH, and nonspecific rabbit IgG. Immunoprecipitation was conducted using anti-p73 antibody and analyzing tyrosine phosphorylation of p73 using Western blotting. When conducting immunofluorescence, cells were grown to 50% confluency on chamber sides. Phospho-Histone and 8-oxoG were stained and the intensities of the fluorescent signal were measured.

Real-time PCR and chromatin immunoprecipitation
RNA was isolated, the total RNA was reverse transcribed with a high capacity cDNA reverse transcription kit, and real-time PCR was used. Chromatin immunoprecipitation was conducted on p73 antibody and a nonspecific rabbit IgG was used as a negative control for HET-1A cells. The p73 binding sites were then analyzed. Certain sequences of primers were used to analyze p73, MUTYH, SMUG1 mRNA levels and ChIP.

PCR array, comet, and abrasic site quantification assays
Comet assays were performed with modifications to the protocol. The cells were mixed with a low melting temperature agarose and applied to a pre-agarose coated slide. A buffer was used to induce cell lysis and then proceeded in electrophoresis under alkaline conditions. The slide was then stained for analysis using CometScore Software. In p73 deficient and the HET-1A cells, the mRNA expressions were compared and analyzed.

Surgical Procedures and immunohistochemistry
Mouse strains that carried the wild type p73 and normal p73 genes were analyzed. Esophagojejunostomy was conducted on these mice that carried the wild type p73 and normal p73 genes. The 15-week mice were euthanized and the lower esophagus was removed for analysis by immunohistochemistry.

Nontumorous esophageal HET-1A cells were treated with a BA mixture and the DNA damage was analyzed by immunofluorescence using phospho-H2A.X and 8-OxoG antibodies. A small exposure to the BA mixture inflicted DNA damage through oxidation and strand breakage. The presence of the p53 family upon BA/A treatment was then analyzed. Despite the presence of DNA damage, there was a lack of increased phosphorylation of p53 at serine 15 to induce p53 activation. P53 was only present when the HET-1A cells were treated with a DNA damaging drug called cisplatin. After BA/A treatment, p53 levels decreased and were undetected after a long duration of time. BA/A treatment of SKGT-4 cells resulted to an up-regulation and activation of p73; the analysis revealed that p73 induction is independent of p53. Because the mRNA levels of p73 were not increased in the epithelial cells, it indicated that the up-regulation occurred at the post-translational stage. The presence of c-Abl protein kinase induces the tyrosine phosphorylation, which then activates the p73 protein.

By inhibiting p73 proteins, increased oxidative DNA damage is apparent. P73 activates SMUG1 and MUTYH that are involved in base excision repair by directly binding to these two glycosylases and regulates its transcription. Therefore, the down regulation of p73 proteins decreases the two glycosylases of SMUG1 and MUTYH[6].